Laboratory of Molecular Biotechnology and Biomaterials, School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea.
Appl Environ Microbiol. 2012 Feb;78(3):684-94. doi: 10.1128/AEM.06274-11. Epub 2011 Nov 18.
7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such as Populus alba and Eucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, an Escherichia coli cell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor, p-coumaric acid, in E. coli for the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feeding p-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate-coenzyme A (CoA) ligase from Petroselinum crispum, chalcone synthase from Petunia hybrida, and chalcone isomerase from Medicago sativa. In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinica were also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase from Arabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase from Streptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction in E. coli, produced 2.7 mg/liter (8.9 μM) 7-OMA upon supplementation with 500 μM p-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 μM) 7-OMA from 500 μM NRN in 24 h.
7-O-甲基橙皮素（7-OMA）是几种植物中重要的黄酮苷元之一，如白杨树和桉树，具有多种药用应用。为了大量生产这种有价值的天然类黄酮，已经开发了大肠杆菌细胞工厂来利用各种植物生物合成途径。在这里，我们首次报道了大肠杆菌中 7-OMA 前体对香豆酸的生成。首先，通过添加对香豆酸并通过引入以下结构基因重建植物生物合成途径来实现柚皮素（NRN）（黄酮）的合成：来自皱叶欧芹的 4-香豆酸-CoA 连接酶、来自杂种矮牵牛的查尔酮合酶和来自紫花苜蓿的查尔酮异构酶。为了增加 7-OMA 的关键前体丙二酰辅酶 A 的可用性，还引入了来自诺卡氏放线菌的酰基辅酶 A 羧化酶 α 和 β 亚基（nfa9890 和 nfa9940）、生物素连接酶（nfa9950）和乙酰辅酶 A 合成酶（nfa3550）的基因。因此，产生的 NRN 在拟南芥的黄酮醇 3-羟化酶的作用下在 3 位羟基化，然后在链霉菌avermitilis 的 7-O-甲基转移酶的存在下在 7 位甲基化生成 7-OMA。二氢山奈酚（DHK）（橙皮素）和樱花素（SKN）作为中间产物产生。在大肠杆菌中过表达黄酮类生物合成和修饰途径的基因，并过量生产丙二酰辅酶 A，在 24 小时内补充 500 μM 对香豆酸时可产生 2.7 mg/L（8.9 μM）的 7-OMA，而仅表达黄酮类修饰酶的菌株可在 24 小时内从 500 μM NRN 中产生 30 mg/L（99.2 μM）的 7-OMA。
